Unusual semi‐extractability as a hallmark of nuclear body‐associated architectural noncoding RNAs

• Published in: The EMBO journal Vol. 36; no. 10; pp. 1447 - 1462
• Main Authors: Chujo, Takeshi, Yamazaki, Tomohiro, Kawaguchi, Tetsuya, Kurosaka, Satoshi, Takumi, Toru, Nakagawa, Shinichi, Hirose, Tetsuro
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 15-05-2017; Blackwell Publishing Ltd; John Wiley and Sons Inc
• Subjects: Animals; Cell Nucleus - chemistry; EMBO22; EMBO32; EMBO36; Extraction; FUS protein; Granular materials; Humans; long noncoding RNA; Molecular biology; Molecular Biology - methods; nuclear body; Nucleoproteins - analysis; Nucleoproteins - isolation & purification; Prion protein; prion‐like domain; Protein Binding; Protein interaction; Proteins; Resource; Ribonucleic acid; RNA; RNA extraction; RNA, Long Noncoding - analysis; RNA, Long Noncoding - isolation & purification; RNA-binding protein; Sequence Analysis, RNA; Shearing; long noncoding RNA; nuclear body; RNA extraction; prion‐like domain; RNA‐binding protein
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201695848

NEAT1_2
long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved
NEAT1_2
extraction by 20‐fold (a property we term “semi‐extractability”), whereas using a conventional method
NEAT1_2
was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50
NEAT1_2
molecules are present in a single paraspeckle. Another architectural lncRNA,
IGS16
, also exhibited similar semi‐extractability. A comparison of RNA‐seq data from needle‐sheared and control samples revealed the existence of multiple semi‐extractable RNAs, many of which were localized in subnuclear granule‐like structures. The semi‐extractability of
NEAT1_2
correlated with its association with paraspeckle proteins and required the prion‐like domain of the RNA‐binding protein FUS. This observation suggests that tenacious RNA–protein and protein–protein interactions, which drive nuclear body formation, are responsible for semi‐extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.
Synopsis
The NEAT1_2 lncRNA – a scaffold for paraspeckle formation – is inefficiently extracted from cells by conventional extraction. A modified RNA extraction protocol allows the identification of several new architectural long noncoding RNAs and sheds light on the properties of cellular RNA‐protein granules.
Many architectural lncRNAs such as NEAT1 are difficult to extract using conventional methods.
An improved cellular RNA extraction method increases yield of architectural, semi‐extractable lncRNAs.
Various semi‐extractable RNAs are identified and found to be localized in subnuclear granule‐like structures.
Semi‐extractability of NEAT1 is dependent on FUS protein, especially its prion‐like domain.
Graphical Abstract
A modified RNA extraction protocol allows the identification of several new architectural long noncoding RNAs and sheds light on the properties of cellular RNA‐protein granules.